A large proportion of naive T lymphocytes respond to cells expressing allogeneic MHC molecules. The magnitude of this reaction underlies the observation that, in the absence of pharmacologically induced immunosuppression, MHC disparate allografts are rapidly rejected. The molecular basis of T cell recognition of allogeneic MHC and, in particular, the role of peptide in this recognition process, is controversial. To investigate this, we have taken advantage of a cell line (T2) that fails to process antigen for presentation by either class I or class II MHC. As a result, T2 transfectants express class II MHC exclusively in the SDS-stable MHC conformer and are therefore mostly peptide-dependent. We also show that MHC molecules on T2 transfectants can be loaded with peptide, converted into the SDS-stable form, and stimulate T cells. Peptide-loaded T2 transfectants are therefore unique reagents that express monomorphic functional complexes of a single class II MHC molecule and a single peptide. In specific aim #1, we will use these cells to determine the percent of alloreactive T cells that are not peptide-specific. This will be accomplished by priming mice with allogeneic, B6, splenocytes and assaying the primed CD4+ T cells for the ability to respond to T2-I-Ab cells that express I-Ab-peptide complexes not expressed by B6 cells. This will be further investigated by cloning T cells with this reactivity pattern and assaying for their responses to T2-I-Ab transfectants loaded with other peptides. In aim #2, we will determine the frequency of alloreactive T cells specific for the most prevalent I-Ab-peptide complexes. Examples of such complexes include Ealpha-peptide-I-Ab and invariant chain peptide-I-Ab. Mice will be primed with B10.A(5R) spleen cells. B10.A(5R) cells express both of these I-A-peptide complexes. The primed T cells will be assayed using B6 spleen cells or methA-I-Ab (an invariant chain negative cell) in the presence or absence of peptide. In aim #3, we will apply these methods to an in vivo allograft system and determine the corresponding frequencies of graft-infiltrating T cells that are either not peptide- specific or specific for these common I-Ab-binding peptides. We will extend these in vivo studies to identify allograft-reactive T cells that are specific for cardiac myocytes. We will then use these T cells to screen HPLC fractions of peptides purified from the I-Ab expressed by myocardial cells. If the myocardial peptides involved in graft rejection are expressed at relatively high levels, we hope to sequence them. Lastly, in aim #4, we will determine if T cells recognize MHC-peptide differently in the context of self MHC vs. allo MHC. We will generate variant Ealpha peptides and compare the dose response curves of allo T cells specific for Ealpha-peptide-I-Ab vs. syngeneic T cells that recognize the same complex. In these same experiments, we hope to identify peptides that can inhibit T cell responses for Ealpha-peptide-I- Ab. Such peptides may be useful in altering the course of in vivo graft rejection.